Cas-OPRAD: a one-pot RPA/PCR CRISPR/Cas12 assay for on-site Phytophthora root rot detection

文献类型： 外文期刊

第一作者： Li, Zhiting

作者： Li, Zhiting;Feng, Wanzhen;Lu, Shengdan;Lin, Mingze;Wang, Zhixin;Chen, Qinghe;Li, Zhiting;Feng, Wanzhen;Lu, Shengdan;Lin, Mingze;Chen, Qinghe;Zhu, Zaobing;Dong, Jiali;Wang, Zhixin;Liu, Fuxiu

作者机构：

关键词： Phytophthora sojae; one-pot RPA/PCR; recombinase polymerase amplification; CRISPR/Cas12a; on-site detection; visual detection

期刊名称：FRONTIERS IN MICROBIOLOGY （ 影响因子：4.0； 五年影响因子：5.1 ）

ISSN：

年卷期： 2024 年 15 卷

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收录情况： SCI

摘要： Phytophthora sojae is a devastating plant pathogen that causes soybean Phytophthora root rot worldwide. Early on-site and accurate detection of the causal pathogen is critical for successful management. In this study, we have developed a novel and specific one-pot RPA/PCR-CRISPR/Cas12 assay for on-site detection (Cas-OPRAD) of Phytophthora root rot (P. sojae). Compared to the traditional RPA/PCR detection methods, the Cas-OPRAD assay has significant detection performance. The Cas-OPRAD platform has excellent specificity to distinguish 33 P. sojae from closely related oomycetes or fungal species. The PCR-Cas12a assay had a consistent detection limit of 100 pg. mu L-1, while the RPA-Cas12a assay achieved a detection limit of 10 pg. mu L-1. Furthermore, the Cas-OPRAD assay was equipped with a lateral flow assay for on-site diagnosis and enabled the visual detection of P. sojae on the infected field soybean samples. This assay provides a simple, efficient, rapid (<1 h), and visual detection platform for diagnosing Phytophthora root rot based on the one-pot CRISPR/Cas12a assay. Our work provides important methods for early and accurate on-site detection of Phytophthora root rot in the field or customs fields.

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